AbstractA method has been developed for the two‐dimensional separation of proteins in a single thin‐layer gel, using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis in the first dimension and isoelectric focusing in the second dimension. After two‐dimensional electrophoresis of concentrated culture supernatants fromStreptococcus mutans, the gels were directly stained with Coomassie Brilliant Blue R‐250 for protein and with periodic acid‐Schiff base reagent for glycoprotein. Glycosyl‐transferases were detected by incubating the gels in a sucrose‐containing buffer, either by visual observation of white spots of the product polysaccharides or by periodic acid‐Schiff staining of the products. All the procedures, including the staining steps, were done in a single gellayer covalently bound to a glass plate. The efficient removal of sodium dodecyl sulfate and renaturation of glycosyltransferases in the gel layers rendered this a simplified and rapid method for the relative molecular mass and isoelectric point determinations in the c